Kim Jinshil, Tai Chin-Hsien, Livingston Natalie K, Patterson-West Jennifer, Stearns Oliver, Son Bokyung, Hinton Deborah M
Gene Expression and Regulation Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Comput Struct Biotechnol J. 2025 May 21;27:2051-2062. doi: 10.1016/j.csbj.2025.05.028. eCollection 2025.
Uncharacterized bacteriophage proteins typically have little homology outside the phage world. An example is the T4 early protein GoF. Although the function of wild type is not known, the GoF mutant (D25Y) affects the level of T4 gene mRNA under certain conditions. To investigate possible GoF functions, we leveraged the power of AlphaFold3. We found that despite having very dissimilar sequences, GoF and 2 other uncharacterized T4 early proteins, MotB.1 and Frd.2, are structurally similar with predicted N-terminal LSm-like fold motifs. Since this motif, which is found throughout biology, is frequently associated with an RNA function and the GoF(D25Y) mutation is found within the predicted LSm-like fold, we hypothesized that these proteins may affect gene expression. Consequently, we used a fluorescent translational reporter system and RT-qPCR to investigate if and how the presence of the proteins affect the expression of an gene placed downstream of the T4 gene 5' untranslated region. We find that the heterologous expression of (D25Y) increases the level of mCherry post-transcriptionally by increasing the stability of the RNA. However, neither WT GoF nor MotB.1 have this effect. We speculate that GoF(D25Y) may represent a gain-of-function mutant that can increase RNA stability. Using AlphaFold3 models we speculate how the D25Y mutation in GoF might facilitate or enhance RNA binding. Our work reveals the power of AlphaFold to find unexpected structure/function relationships among uncharacterized proteins.
未表征的噬菌体蛋白通常在噬菌体世界之外几乎没有同源性。一个例子是T4早期蛋白GoF。尽管野生型的功能尚不清楚，但GoF突变体（D25Y）在某些条件下会影响T4基因mRNA的水平。为了研究GoF可能的功能，我们利用了AlphaFold3的强大功能。我们发现，尽管GoF与另外两种未表征的T4早期蛋白MotB.1和Frd.2的序列非常不同，但它们在结构上相似，具有预测的N端LSm样折叠基序。由于这种在整个生物学中都存在的基序经常与RNA功能相关，并且在预测的LSm样折叠中发现了GoF（D25Y）突变，我们推测这些蛋白质可能会影响基因表达。因此，我们使用荧光翻译报告系统和RT-qPCR来研究这些蛋白质的存在是否以及如何影响位于T4基因5'非翻译区下游的一个基因的表达。我们发现（D25Y）的异源表达通过增加RNA的稳定性在转录后增加了mCherry的水平。然而，野生型GoF和MotB.1都没有这种作用。我们推测GoF（D25Y）可能代表一个功能获得性突变体，可以增加RNA稳定性。利用AlphaFold3模型，我们推测GoF中的D25Y突变可能如何促进或增强RNA结合。我们的工作揭示了AlphaFold在发现未表征蛋白质之间意外的结构/功能关系方面的强大功能。
